The purpose of the proposed research is to identify, purify and characterize two nucleotide binding proteins present in platelets important for regulation of platelet activation and inhibition. One of these proteins is located on the outside of the intact platelet membrane. This polypeptide interacts with ADP and ATP, plays a role in the modulation of platelet shape change and may represent the putative ADP receptor. The approach is to label covalently the membrane polypeptide with a radioactive analog of the nucleotide containing a alkylating group (affinity reagent). The labelled polypeptide will be purified and the peptides and/or amino acids responsible for binding will be identified. The functional consequences of the inhibition of the binding protein with respect to fibrinogen binding and cyclic AMP concentrations will be determined. The second proteins is cyclic nucleotide phosphodiesterase (s) enzymes involved in the regulation of the cyclic AMP (and cyclic GMP) of platelets and hence the responsiveness to many agonists. The subcellular localization of these enzymes will be determined and the enzymes purified to homogenity. A cyclic nucleotide affinity reagent will be used to establish the kinetics of the inactivation and to localize the binding site within the enzyme.